1. Field of the Invention
The present invention relates to instruments for the analysis of particles in a fluid, and their use.
2. Description of Related Art
Flow-type particle analyzers, such as flow cytometers, are well known analytical tools that enable the characterization of particles on the basis of optical parameters such as light scatter and fluorescence, or by electrical properties, such a impedance. In a flow cytometer, for example, particles, such as molecules, analyte-bound beads, or individual cells, in a fluid suspension are passed by a detection region in which the particles are exposed to an excitation light, typically from one or more lasers, and the light scattering and fluorescence properties of the particles are measured. Particles or components thereof typically are labeled with fluorescent dyes to facilitate detection, and a multiplicity of different particles or components may be simultaneously detected by using spectrally distinct fluorescent dyes to label the different particles or components. Typically, detection is carried out using a multiplicity of photodetectors, one for each distinct dye to be detected. Both flow and scanning cytometers are commercially available from, for example, BD Biosciences (San Jose, Calif.). A description of flow cytometers is provided in Shapiro, 2003, Practical Flow Cytometry, 4th ed. (John Wiley and Sons, Inc. Hoboken, N.J.), and in the references cited therein, all incorporated herein by reference.
In a typical flow cytometer, the particle-containing sample fluid is surrounded by a particle-free sheath fluid that forms an annular flow coaxial with the sample fluid as is passes through the detection region, thereby creating a hydrodynamically focused flow of particle-containing sample fluid in the center of the fluid stream, surrounded by particle-free sheath fluid. Typically, the ratio of sheath fluid to sample fluid is high, with the sample fluid forming only a small fraction of the total fluid flow through the detection region.
Typically, flow cytometer systems have been implemented using pressure-driven fluidics in which the sample and sheath fluid are provided to a flow cell, which contains the detection region, under a pressure greater than ambient pressure. Changes in the flow rate through the flow cell of a pressure-driven fluidics system are achieved by varying the pressure in the sample tube and/or the sheath fluid reservoir that feed into the flow cell. The ratio of sample fluid to sheath fluid flowing through the flow cell is governed both by the pressure levels in the sample tube and sheath fluid reservoir, and by the ratio of the resistances of the sample fluid and sheath fluid paths.
Alternatively, flow cytometer systems have been implemented using vacuum-driven fluidics in which a vacuum pump draws a vacuum downstream of the flow cell, and the sample and sheath fluids are held at ambient pressure. Changes in the flow rate through the flow cell of a vacuum-driven fluidics system are achieved by varying the vacuum drawn by the vacuum pump, and the ratio of sample fluid to sheath fluid flowing through the flow cell is governed by the ratio of the resistances of sample fluid and sheath fluid paths.
In general, the design of pressure-driven fluidics systems is more complicated than is the design of vacuum-driven fluidics systems because of the need for components, including tubing, connections, and seals, that withstand high system pressure levels. In contrast, in a vacuum-driven flow cytometry system, the design of the fluidic connections to the sample tube and sheath fluid supply reservoir is greatly simplified as it does not require the use of pressurizable tubings, connections, and seals. The elimination of pressurized sample tubes further facilitates the design of auxiliary equipment, such as automatic tube lifters and robotic sample loaders.
U.S. Pat. No. 5,395,588, incorporated herein by reference, describes a vacuum control system for use in a flow cytometer. The system includes a vacuum pump that pulls a sheath fluid from an open supply reservoir through a flow cell wherein cell analysis occurs, and discharges the flow cell effluent to an open waste reservoir. A pressure drop is created through the conduit leading from the supply reservoir to the flow cell, which also aspirates a sample consisting of a particle (e.g., cell) suspension from an open sample vessel into and through the flow cell. The flow rate of the system is regulated by monitoring the vacuum level at the outlet of the flow cell. A control circuit coupled to the vacuum sensor adjusts the electric power applied to the vacuum pump motor to maintain a predetermined vacuum level at the outlet of the flow cell.